Résumé - L’ADNr 5S chez le bivalve Cerastoderma edule : séquence nucléotidiquede l’unité de répétition et localisation chromosomique par rapport à l’ADNr 18S-28S. L’unité de répétition complète de l’ADNr 5S a été amplifiée par PCR chez lebivalve Cerastoderma edule et plusieurs clones ont été séquencés. En outre, le produitde PCR de plusieurs individus a été digéré par des enzymes de restriction. Les résultatsobtenus indiquent que l’ADNr 5S est organisé sous forme de répétitions en tandemdont l’unité mesure 544-546 pb, parmi lesquelles 120 pourraient représenter la région
genes d’ARNr 5S / espaceur non transcrit / Cerastoderma edule / FISH
1. INTRODUCTION
The 5S ribosomal DNAs (5S rDNA) of many eukaryotes has been clonedand characterized. In most cases, it is organized as clusters of tandem repeatsof several hundred base pairs (bp), consisting of a coding region and a non-transcribed spacer region !14!. Accumulated data demonstrate that, while thecoding region is highly conserved among taxa, both with respect to lengthand nucleotide sequence, the spacer region evolves more rapidly and can showvariation both within and between species (e.g. [7, 19!).
In addition to the gene encoding the 5S rRNA, many species contain genevariants and pseudogenes, differing from the gene by a variable number of sub-stitutions and deletions [5, 18, 24!. Moreover, it has been well documented thatXenopus laevis has three types of 5S rDNA sequences with developmentallyregulated expression !32]. More than one type of 5S rDNA sequence with dif-ferential expression was also seen in the chicken [13] and some fish [12]. Asis true for several other families of tandemly repeated genes, the 5S rDNArepeats evolve concertedly !3!, i.e. in intra-specific comparisons a high degreeof sequence similarity is usually observed between independent repeats. Thus,5S rDNA sequences are regarded as potentially useful in revealing phylogeneticrelationships.
In contrast to genes encoding 18S, 5.8S and 28S rRNA (18S-28S rDNA),where chromosomal location can be determined by selective staining of nucleo-lus organizer regions and in situ hybridization, 5S rDNA can only be detectedby in situ hybridization. This could explain the fact that, in general, there isless information available on the chromosomal location of 5S rDNA. In bivalvemolluscs, very little attention has been paid to 5S rDNA. To date, only the5S rRNA of three species belonging to different subclasses has been sequenced:Solemya velum (Protobranchia), Calyptogena magnifica (Heterodonta) [25] andMytilus edulis (Pteriomorphia) !6!. The chromosomal location of 5S rDNA wasdetermined in a pectinid species, Aequipecten opercularis !10!.
This work provides for the first time the nucleotide sequence of the whole5S rDNA repeated unit of a bivalve species, the cockle Cerastoderma edule(Heterodonta, Cardiidae), and an analysis of the intra- and inter-individualvariation. In addition, it reports the chromosomal location of 5S rDNA and itsphysical relation to 18S-28S rDNA.
2. MATERIALS AND METHODS
Specimens of C. edule were collected from several locations (Pontevedra,Vilanova de Arousa, Ponteceso, Ria do Burgo and Cedeira) along the Galiciancoast (NW Spain). Genomic DNA was extracted from muscle tissue accordingto Winnepenninckx et al. !31!.
2.1. PCR amplification, cloning and sequencing
The amplification mixture used for PCR (50 OL) contained 500 ng of ge-nomic template DNA, 1 vM each primer, 250 vM dNTPs, 1.25 U of Taq poly-merase (Boehringer Mannheim) and the buffer recommended by polymerasesuppliers. The primers were 5’-CAACGTGATATGGTCGTAGAC-3’ (A) and5’-AACACCGGTTCTCGTCCGATC-3’ (B), obtained from the 5S rRNA se-quence of the mussel M. edulis [6], and 5’-CAAGCACAGAGGCAGGAG-3’(C) and 5’-CGATCCGCGGTTTACCTG-3’ (D) obtained from the C. edule5S rDNA spacer region. Thirty standard PCR amplification cycles were per-formed at an annealing temperature of 64 °C with primers A and B and56 °C with primers C and D. The PCR product generated with both sets ofprimers was purified using Geneclean (BIO 101, INC), ligated into the plasmidpGEM-T Easy, using pGEM-T Easy Vector System II (Promega), and subse-quently transformed into E. coli JM109 cells. Recombinant clones were selectedas white colonies on ampicillin plates containing X-gal and IPTG. PlasmidDNA purification of four clones (two with insert obtained with primers A andB, and the other two with insert obtained with primers C and D) was car-ried out as described by Sambrook et al. [23]. Both strands of each clone weresequenced by the dideoxy-sequencing method with the AutoRead kit (Phar-macia). Automatic sequencing was performed on an A.L.F. express sequencer(Pharmacia). Sequences were aligned using CLUSTAL V with both fixed andfloating gap penalties of 10 !9!. The nucleotide sequences have been depositedin the EMBL DNA data base under the accession numbers AJ132196-132199.
2.2. Chromosome preparation and FISH
Metaphases were obtained from gill cells following the procedure describedby Thiriot-(!uievreux and Ayraud !28!. A specific probe of C. edule 5S rDNAwas produced by PCR using the primers A and B. Labelling was obtainedusing the PCR procedure described above, but with a different dNTP con-centration (100 vM dATP, 100 wM dCTP, 100 OM dGTP, 160 RM dTTP and35 vM digoxigenin-11-dUTP). A recombinant plasmid containing 185, 5.8S and28S genes plus intergenic spacers of Drosophila melanogaster was used as probeto localize 18S-28S rDNA. After extraction by alkaline lysis (23], the wholeplasmid was labelled with digoxigenin-11-dUTP employing the BoehringerMannheim nick translation kit. FISH was carried out as in Insua et al. !10!,but the post-hybridization washing was carried out with a 65 % formamidesolution in the case of 5S rDNA. C-banding was performed according to themethod of Sumner [26] but slides were stained with acridine orange followingMartinez-Lage et al. [15]. The examination of chromosome spreads was per-formed with a Nikon fluorescence photomicroscope equipped with appropriatefilters and photographs were taken with Kodak Ektachrome film.
3. RESULTS
The 5S rDNA repeat unit of C. edule was amplified by PCR using primers Aand B, designed from the 5S rRNA sequence of M. edulis (6!. PCR amplificationproduced a single band of approximately 550 bp. This amplification productwas cloned and then two clones were sequenced (Cel and Ce2). Two additionalprimers, C and D, derived from the spacer region of the just sequenced C. edule5S rDNA, were also used to produce a new PCR amplification of the 5S rDNArepeat unit. A single band of 550 bp was also obtained, and after cloning twoclones were sequenced (Ce3 and Ce4).
The complete repeat unit consists of 544-546 bp and the alignment of full-length sequences of the four clones consists of 548 bp (figure 1). Comparisonwith available bivalve sequences [6, 25] allows us to infer that the coding regionstarts 5’ with GTC and ends with CTT to give a 5S rRNA size of 120 nucleotides(figure !). However, the 5S rRNA from mussel M. edulis ends with ACA andhas a size of 119 nucleotides (6!. Therefore, the assignment of the 3’ end andthe 5S rRNA size must be considered tentative. The inferred coding regionof the C. edule 5S rDNA is invariable between clones, ignoring the sequencecorresponding to primer A in clones Cel and Ce2.
Several sequences of the eukaryotic 5S rDNA involved in the transcriptioncan be identified in the sequences obtained from C. edule: internal control
region [22]; sequence elements related to upstream regulatory regions of thecoding region as TATATA [17]; and terminator sequences composed of fourthymidine residues [1] located downstream of the coding region (figure 1). TheG/C content, determined in the consensus sequence of the four clones, is higherin the coding region (54.2 %) than in the spacer region (45.8 %).
The spacer region showed some variation, ranging from 424 to 426 bp inlength. Eight variable sites were detected in the alignment, four of whichcorrespond to gaps and four to nucleotide substitutions (figure 1). Nevertheless,three of the clones (Cel, Ce2 and Ce3) are almost identical, only two gapsassociated with a run of thymidine residues at the 5’ end being observed. Tofurther examine the extent of the variation, the 5S rDNA was amplified withprimers A and B from nine additional individuals collected along the Galiciancoast. The product obtained was digested with the enzymes Alu I, Hae III,Rsa I and Taq I. No variation was found in the restriction pattern generated bythese enzymes, except in one individual which showed intra-individual variationconcerning the Rsa I restriction pattern. This had three bands as is to be
expected from the sequences determined here, but also an additional band,resulting from the absence of one enzyme target in the spacer region.
Comparison of the 5S rDNA coding sequence of C. edule with previouslypublished sequences of other bivalve species (figure 2) reveals no differenceswith Calyptogena magnifica (Heterodonta, Vesicomyidae), and four nucleotidedifferences with Solemya velum (Protobranchia, Solemyidae) and Mytilus edulis(Pteriomorphia, Mytilidae).
The chromosomal location of the 5S rDNA was determined by FISH, usinga specific probe obtained by PCR. Forty-one metaphases, belonging to fourindividuals, were analysed. The pattern most frequently observed displays atotal of nine hybridization sites distributed on the telomere of the long armsof five chromosome pairs (figure 3a). Since most chromosomes in the karyotype
of C. edule are submetacentric (12 pairs), and the remaining chromosomes aresubtelocentric (4 pairs) or telocentric (3 pairs), with small differences in size[11], identification of chromosome pairs carrying 5S rDNA cannot be accuratelydetermined.
To establish the relative position of 5S rDNA and 18S-28S rDNA, FISH wascarried out with a heterologous 18S-28S rDNA probe. Forty-seven metaphasesfrom four individuals were examined. In all cases, hybridization signals werefound spread along the short arms of one pair of chromosomes characterizedby having short arms of different size between homologous chromosomes (fig-ure 3b). After performing C-banding, this pair showed constitutive heterochro-matin regions of unequal size between homologous chromosomes (figure 3c).Propidium iodide staining was less intense at these heterochromatin regions,especially at the largest one. This made it possible to recognize them afterFISH (figure 3a). Hybridization signals with 5S rDNA probe were not observed
in these heterochromatin regions (figure 3a) indicating that 5S and 18S-28SrDNA are not linked.
4. DISCUSSION
This is the first report on the characterization of the whole 5S rDNA repeatunit in a bivalve species. The results obtained suggest that C. edule 5S rDNAexhibits the conventional tandem arrangement. Evidence is provided by the factthat the PCR amplification of the 5S rDNA unit was obtained using contiguousprimers located in opposite orientation. The length of the inferred 5S gene is120 bp and the spacer region ranges between 424 and 426 bp. Totalling 544-546 bp, this size is intermediate between other 5S rDNA repeat units observedin invertebrates such as the 300-450 bp of some Diptera [8] and the 589 bp ofa crustacean species !20!.
The four sequenced clones of C. edule display an identical coding regionand potential regulatory elements are identifiable in all of them. Therefore, theoccurrence of gene variants or pseudogenes among the repeat units analysedhere can be considered improbable.
In the spacer region of 5S rDNA, three of the clones analysed are almostidentical as only two variable sites were found and these appear to result fromreduction/expansion of the thymidine-rich region at the 5’ end. Excluding thevariable sites associated with these thymidine regions, the fourth clone displaysdifferences at five nucleotide positions. As the clones analysed were obtainedby PCR, it cannot be excluded that some of the nucleotide variations weredue to Taq polymerase misincorporation. The occurrence of this minimal intra-individual variation and the identification of the same restriction patterns afterenzyme digestion in the individuals examined suggest that 5S rDNA would bean appropriate region for assessing the relationships between C. edule and otherbivalves.
Comparison of the 5S rDNA coding sequence of C. edule and the threeavailable sequences of bivalves shows little interspecific variation. C. edule andCalyptogena magnifica, two species whose superfamilies are considered to have acommon ancestor in Late Paleozoic !16!, share the same sequence. On the otherhand, C. edule differs from S. velum and from M. edulis at only four nucleotidepositions, although C. edule and S. velum share a common ancestor in Pre-cambrian and C. edule and M. edulis in Early Paleozoic !16!. This might indicatethat phylogenetic information provided by the coding sequence may be limited,but a sufficiently large species sample and comparison of the supposedly fast-evolving spacer sequences could establish a utility of 5S rDNA for phylogenyestimations.
FISH revealed a total of nine hybridization sites. Thus, one of the pairs seemsto bear 5S rDNA in heterozygosity; that is, it is absent in one chromosome or ispresent in such a low copy number that it is not sufficient for detection by FISH.Since chromosome pairs cannot be distinguished unambiguously after propid-ium iodide counterstaining it is not possible to determine if it is always thesame pair that shows 5S rDNA in heterozygosity. Differences between homol-ogous chromosomes concerning the size of the 5S rDNA hybridization signalswere observed in amphibians [30] and this type of variation is not unusual inthe case of repetitive sequences located at telomeres. Also, 18S-28S rDNA and
heterochromatin regions identified here in C. edule display different sizes. Sisterchromatid exchanges and unequal meiotic crossing-over events could cause thesize of rDNA clusters to fluctuate randomly so that heterozygosity of the rDNAclusters is the rule, and homozygosity the exception.
The distribution of 5S rDNA in C. edule is very different from that found inthe bivalve analysed so far, A. opercudaris. In this pectinid species, 5S rDNAwas identified at two sites of one arm of a metacentric pair (10!. The differencesbetween these two species contrast, for example, with the tendency of 5S rDNAin mammals to be localized in the terminal region of a pair of chromosomes(27!. However, they are similar to those found in other groups such as anuranamphibians where both number as well as position can be very different betweenspecies (30!.
Unlike the 5S rDNA, the location of 18S-28S rDNA in C. edule is restrictedto short arms of one chromosome pair, as was already observed in individualsof C. glaucum populations using silver staining (29!. Both types of rDNA havebeen found to be linked in several animals and plants [2, 4, 21!, nevertheless,the most common situation in higher eukaryotes is the absence of linkage. Theresults obtained in this work show that this also occurs in C. edule.
ACKNOWLEDGEMENT
This work has been supported by project XUGA 10302B97 of the GalicianGovernment.
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